Successful placental development has long-term implications for adult health, is necessary for embryo survival and proper fetal growth, and is dependent on vascularization. During pregnancy, placental blood vessels elongate, dilate, and extend new sprouts to facilitate maximal transfer of nutrients from maternal to placental vasculatures for hematotrophic support of the developing embryo/fetus. The long-term research goal is to identify and determine the physiological pathways that promote vessel growth within the placenta. Endothelial Progenitor Cells (EPCs) reside in the bone marrow, migrate, and incorporate into growing blood vessels to form new endothelial cells. A firm relationship has been established between EPCs and pulmonary hypertension, cardiovascular risk, ischemia, atherosclerosis, and pregnancy. EPC numbers decrease in preeclampsia and gestational diabetes, and maternal insulin therapy increases EPC numbers during diabetic pregnancy. We have isolated and characterized EPCs from the peripheral blood of newborn pigs. These EPCs express integrins, and use these transmembrane receptors to adhere and migrate in vitro on osteopontin (OPN) an extracellular matrix molecule with prominent expression in the placenta of both humans and pigs. When EPCs are cultured alone as a monolayer on the surface of 3D collagen matrices supplemented with angiogenic factors, they fail to invade the matrix. However, if cultured with adult human umbilical vein endothelial cells (HUVECs), which invade the matrix and form vascular structures, the EPCs incorporate into these vascular structures. This is similar to their behavior in vivo. Importantly, OPN dose-dependently increases the number of EPCs that incorporate into these vascular structures. The central hypothesis is that OPN recruits EPCs to sites of neovascularization within the placenta. Once there, EPCs communicate directly with adult endothelial cells via junctional adhesion molecules (i.e., gap, tight and adherens junctions) to incorporate into growing vessels. The two objectives are: (1) Determine the intercellular signals that allow EPCs to incorporate into established vasculature; and (2) Determine whether EPCs incorporate into placental vascular networks pigs. Completion of these objectives will aid the rational design and development of novel intravenous EPC-based therapies to normalize placental vasculature, and lessen effects of preeclampsia, gestational diabetes, and other gestational diseases. PUBLIC HEALTH RELEVANCE: Failed pregnancies elicit significant social and economic burdens; therefore, a complete understanding of these events is warranted. This proposal will significantly advance our understanding of how populations of cells found in the blood contribute to new blood vessel growth, which is a required step in pregnancy. Such knowledge will ultimately aid to decrease the socio-economic burdens associated with aberrations or deficiencies in the process.